Influenza A infects a wide range of avian and mammalian hosts. The constant ability of the virus to evolve requires reformulation of seasonal influenza vaccines on a yearly basis. The virus has eight genomic RNA segments; reassortment of genomic RNAs from different strains and subtypes of influenza A is responsible for sporadic emergence of pandemic flu (Palese, P.; Shaw, M. L. Orthomyxoviridae: The Viruses and Their Replication. In Fields Virology, 5th ed., 2001; and Knipe, D. M., Howley, P. M., Eds.; Lippincott Williams & Wilkins: Philadelphia, Pa., 2007; Vol. 2, pp 1647-1689). Alternatively, all eight genomic RNAs may be derived from an avian virus, and such a progenitor virus then undergoes multiple mutations in the process of adapting to a mammalian host (Taubenberger et al., Nature. 2005; 437(7060): 889-93).
Antivirals are used for both prophylactic and therapeutic treatments of influenza infection. The available treatment options for influenza are limited. Current antivirals are directed against the M2 ion-channel protein (adamantanes) and neuraminidase (zanamivir and oseltamivir). The adamantine drugs, amantadane and rimantadine, are ineffective due to emergence of resistance (predominantly through a M2 mutation, S31N) and these drugs, in general, are not in clinical use. The neuraminidase (NA)-inhibiting oral drug, oseltamivir (Tamiflu) is widely used for treating flu. Oseltamivir-resistant seasonal influenza A strains have been circulating for several years (Moscona, N Engl J Med. 2005; 353(25):2633-6). The mutant viruses predominantly contain the NA H274Y mutation. When accompanied by compensatory mutations, the mutant viruses exhibit fitness comparable to wild-type influenza A and remain resistant to oseltamivir (Bloom et al., Science. 2010; 328(5983):1272-5). These mutations can emerge in almost all influenza A subtypes/strains, including the pandemic 2009 H1N1 virus (Memoli et al., J Infect Dis. 2011; 203(3):348-57), resulting in a major concern for an effective treatment of flu. Therefore, new drugs are essential for treating drug-resistant and future pandemic flu strains.
Influenza A contains eight negative-stranded RNA genomic segments. The three largest genomic RNA segments encode the viral RNA-dependent RNA polymerase (RdRP) proteins consisting of the polymerase acidic protein (PA) and polymerase basic protein 1 (PB1) and 2 (PB2) subunits. The PA subunit: (i) has endonuclease activity (ii) is involved in viral RNA (vRNA)/complementary RNA (cRNA) promoter binding, and (iii) interacts with the PB1 subunit (reviewed by Das et al., Nat Struct Mol Biol. 2010; 17(5):530-8). PA has two domains, PAN (a ˜25 kDa N-terminal domain; residues 1-197) and PAC (˜55 kDa C-terminal domain; residues 239-716). Crystal structures of PAC have been determined in complexes with N-terminal fragments of PB1 (He et al., Nature. 2008; 454(7208):1123-6).
The RdRP of influenza A is responsible for the replication and transcription of the viral RNA genes. The viral mRNA transcription involves a cap-snatching mechanism in which the polymerase binds to cellular mRNA via the 5′-cap and cleaves the mRNA 12-13 nucleotides downstream. The cleaved RNA fragment containing the 5′ cap acts as a primer for viral mRNA synthesis (Plotch et al., Cell. 1981; 23(2):847-58). Cap-snatching is an important event in the life cycle of all members of the Orthomyxoviridae family including influenza A, B and C viruses, and the host cell has no analogous activity. Therefore, inhibitors of cap-snatching would act against all influenza subtypes and strains, including tamiflu-resistant influenza A viruses, and will not interfere with host cell activities.
The complete structure of the viral polymerase has not yet been determined at atomic resolution; however, recent structural studies of parts of the influenza A polymerase (reviewed by Das et al., Nat Struct Mol Biol. 2010; 17(5):530-8) have begun to elucidate the architecture of this complex and started to identify multiple promising target sites for designing new influenza drugs. The crystal structures of the N-terminal domain of PA subunit (PAN) from H5N1 (Yuan et al., Nature. 2009; 458(7240):909-13) and H3N2 (Dias et al., Nature. 2009; 458(7240):914-8) viruses established that the PAN domain contains the endonuclease active site composed of conserved acidic residues E80, D108, and E119 positioned in a deep cleft. Blocking the binding of host mRNAs to the cleft and/or inhibiting the cleavage of the host mRNAs would inhibit the synthesis of the viral mRNAs and thereby, inhibit replication of influenza A.
The PAN domain of 2009 pandemic H1N1 virus polymerase (residues 1-204) has now been crystallized in three distinct forms (U.S. patent application Ser. No. 13/554,709). These new crystal forms provide for the determination of 3-dimensional structures of PAN with endonuclease inhibitors. In addition, a high-throughput methodology (U.S. patent application Ser. No. 13/554,709) has been developed and optimized for screening compounds to inhibit influenza endonuclease. Additional crystal forms of PAN, suitable for structure based drug design, have recently been reported by Kowalinski et al. (PLOS Pathogens. 2012; 8(8):e1002831) using a 2009 pandemic H1N1 sequence and by Dubois et al. (PLOS Pathogens. 2012; 8(8):e1002830) using a A/goose/Guangdong/1/96 (H5N1) sequence.
Compounds that inhibit influenza endonuclease may have inhibitory effects on other drug targets owing to the conserved geometry of the catalytic metals in nucleases and polynucleotidyl transferases. Indeed, early influenza endonuclease inhibitors were developed into an anti-AIDS drug targeting HIV-1 integrase (Summa et al., J Med Chem. 2008; 51(18):5843-55). Other viral drug targets with similar geometry at their catalytic cores include but are not limited to: NS5b RNA-dependent RNA polymerase of hepatitis C virus (Summa et al., J Med Chem. 2008; 51(18):5843-55), RNase H of HIV-1 reverse transcriptase (Himmel et al., Structure. 2009; 17(12):1625-35), herpes virus terminase (Nadal et al., Proc Natl Acad Sci USA. 2010; 107(37):16078-83), and SARS coronavirus NTPase/helicase. Two metal chelating compounds have also been found to have antibacterial effects (Drakulić et al., Chem Med Chem. 2009; 4(12):1971-75) and inhibit bacterial prenyl transferases specifically (Zhang et al., ACS Med Chem Lett. 2012; 3(5):402-6). In addition to having antiviral and antibacterial effects, two metal chelating agents can have cytotoxic effects on eukaryotic cells. One set of compounds was found to have selective anti-leukemic cytotoxicity by inhibiting a terminal deoxyribonucleotidyl transferase (Locatelli et al., Mol Pharm. 2005; 68(2):538-50). In addition, it has been suggested that administration of D-serine with a D-amino acid oxidase (DAAO) inhibitor could allow for more effective delivery of D-serine to the brain, which could be effective in the treatment of symptoms of schizophrenia. Several compounds related to 3-hydroxypyridin(1H)2-ones and its aza-analogs have recently been reported to have activity as D-amio acid oxidase inhibitors (Hondo, et al., J. Med. Chem. 2012, 56, 3582-3592; Duplantier et al, J. Med. Chem., 2009, 52, 3576-3585).